A transgenic mouse, which is produced by introducing a gene-carrying vector, has been widely used to utilize a gene of interest and its expression product. Unfortunately, in conventional transgenic mice, a transgene is introduced into any site at random, and the positional effect of the insertion site may cause reduced expression of the transgene. In addition, conventional gene transfer methods can not control the copy number of a transgene, and limit the size of a transgene to about 200 kb. Due to these problems, it was difficult to clone a gene or gene cluster of more than 200 kb which is not uncommon to mammalian genes, optionally comprising a regulatory region, into a vector. In the conventional gene transfer methods, intrinsic functions of a transgene could not thus be reconstituted and examined, which situation has set a limit.
In the presence of such a problem, the present inventors have developed a technique for producing a transchromosomic mouse by using a novel chromosomal transfer method that introduces genes at a chromosomal level (Non-Patent Literature 1). This technique has allowed a human chromosome or a fragment thereof to be introduced into a mouse embryonic stem (ES) cell, whereby chimeric mice have been produced. This study demonstrated that the human chromosome fragment has been independently retained in ES cells; a plurality of human genes have been expressed in a tissue-specific manner; and some human chromosomes have been able to be partially transmitted to offspring after having undergone meiosis. The present inventors also have introduced the entire human chromosome 21 (about 35 Mb) into a mouse, and have created a Down syndrome model mouse having a high practical value (Non-Patent Literature 2). Analysis of this mouse has revealed effectiveness of the chromosome vector because the mouse has exhibited the physiological expression pattern of the genes of the introduced human chromosome 21.
The techniques the present inventors have employed further include chromosome engineering procedures such as a chromosome deletion method using a telomere truncation technique utilizing an artificial telomere sequence and a chromosome cloning method using a Cre/loxP system. These methods have allowed for construction of a human artificial chromosome containing only a target region. As a result, the present inventors have successfully constructed a human artificial chromosome (HAC) vector containing a specific human chromosome region having a size of mega bases (Mb), and have demonstrated that the vector functions in a mouse individual (Non-Patent Literature 3). Furthermore, the above techniques have been used to construct a novel HAC vector without known genes (Non-Patent Literature 4). In addition, based on the above background, the present inventors have successfully achieved stable expression of a gene of interest by introducing into any cell a HAC vector carrying the gene of interest. Additional examples of a humanized model mouse carrying the HAC vector have been produced as follows: a drug-metabolizing enzyme CYP3A gene cluster (1 Mb) of human chromosome 7 and a human DMD gene (2.5 Mb) responsible for human X-linked muscular dystrophy have been each cloned into a HAC vector (CYP3A-HAC, DMD-HAC); and these vectors have been each introduced into a mouse ES cell to produce mice (Patent Literature 1, Non-Patent Literature 5).
A tissue retention rate and expression analysis of the mouse having the CYP3A-HAC have demonstrated that the CYP3A gene cluster on the HAC has been retained in each tissue of the mouse (FIG. 8, Patent Literature 1). Its expression pattern has been similar to that of a human tissue counterpart. That is, the expression pattern has been specific to a liver and a small intestine. In addition, a tissue retention rate and expression analysis of the mouse having the DMD-HAC have demonstrated that the DMD-HAC has been retained in each tissue of the mouse (FIG. 4A, Non-Patent Literature 5). The mouse has expressed, like a human, at least three splicing isoforms known to be expressed in a tissue-specific manner in a human. This series of results suggest usefulness of the HAC-carrying mouse as a novel gene (gene group)-transfer alternative for a conventional transgenic mouse.
Mammalian artificial chromosome vectors, including a human artificial chromosome, have advantages that conventional vector systems (e.g., a virus, a YAC, a BAC, a PAC, a cosmid, and a plasmid) do not have. Thus, the mammalian artificial chromosome vectors should be useful as a system for analyzing functions of a novel gene and for generating a humanized model animal. For example, Patent Literatures 2 and 3 disclose HAC vectors in which human chromosome 14 or 21 was modified; the chromosome was reduced in size to yield a fragment; and the fragment was relatively stably retained in cells.
Unfortunately, with regard to the human chromosome 21 transferred mouse (i.e., a Down syndrome model mouse) or the HAC vector transferred mouse, which enables the introduction of a gene of Mb units that was impossible for conventional genetically modified mice, there exist at least the following problems: that is, the human chromosome vector has a decreased retention rate; the retention rate varies among tissues and individuals; and the frequency of transmission to offspring is not stable. This facilitates the need to always consider the retention rate of the HAC vectors. Further, when the involvement with functions of a specific gene region or diseases is studied, there is a case where it may be difficult to analyze in detail and precisely a mode of expression of a gene of interest and its expression product at a tissue and/or cellular level. These things will constitute a barrier to highly reproducible, uniform analysis.
Moreover, in the case of conducting the cell fusion between a mouse cell and a human cell, a human chromosome is known to be unstable in the mouse cell. Because the human chromosome, including a human artificial chromosome vector, has thus a variable retention rate in the mouse cell, when the human artificial chromosome vector is introduced into a mouse cell to generate a transgenic mouse, the human artificial chromosome vector does not exhibit full advantages as an artificial chromosome vector. When a mouse cell having a transgene or a transgenic mouse is generated, the retention rate of the transgene should be improved and be made constant. This can promise more detailed, precise, highly reproducible gene function analysis or effective recovery of the expression product of the transgene.